A: Bid (22 kDa) was not cleaved (expected size of 15 kDa) in K562 cells in untreated control cells (lane 1), treated with 5 mM (lane 2) and 10 mM (lane 3) 31 benzamide for 20 h as detected by western blot analysis using anti-BID (lower panel) and anti-beta actin (upper panel) antibody. B: Release of cytochrome c from mitochondria to cytoplasm. The cytoplasmic extract of untreated control K562 cells and cells treated with different concentrations of benzamide for 24 h were separated, transferred and probed with anti-cytochrome-c (lower panel, 13 kDa) and anti-beta actin (upper panel, 42 kDa), as described in methods. Lane 1 represents result in untreated control cells, while lanes 2 and 3 represent result with the cells treated with 5 mM and 10 mM benzamide, respectively. C: Translocation of AIF from mitochondria to nucleus in K562 cells. K562 cells after treatment with benzamide for 24 h as well as untreated cells were fractionated into mitochnodrial, cytoplasmic, nuclear fraction and the separated proteins were transferred, probed with anti-AIF antibody and labeled as AIF (mito), AIF (cyto) and AIF (nuc), respectively. Anti-beta actin (labeled as actin) and anti-histone 2B (labeled as H2B) were used to show equal loading for cytoplasmic and nuclear fraction. Lanes 1, 2 & 3 represent result obtained with untreated control, cells treated with 5 mM and 10 mM BA, respectively.
